DNA encoding Erwinia amylovora hypersensitive response elicitor and its use

ABSTRACT

The present invention is directed to an isolated DNA molecule from  Erwinia amylovora  that encodes a protein or polypeptide which elicits a hyersensitive response in plants. This isolated DNA molecule can used to impart disease resistance to plants, to enhance plant growth, and/or to control insects on plants. Plants or plant seeds transformed with a DNA molecule encoding a hypersensitive response elicitor protein or polypeptide can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.

This application is a division of U.S. patent application Ser. No.09/120,927, filed Jul. 22, 1998 and now U.S. Pat. No. 6,262,018 claimsbenefit of U.S. Provisional Patent Application Ser. No. 60/055,108,filed Aug. 6, 1997.

FIELD OF THE INVENTION

The present invention relates to a hypersensitive response elicitor fromErwinia amylovora and its use.

BACKGROUND OF THE INVENTION

Interactions between bacterial pathogens and their plant hosts generallyfall into two categories: (1) compatible (pathogen-host), leading tointercellular bacterial growth, symptom development, and diseasedevelopment in the host plant; and (2) incompatible (pathogen-nonhost),resulting in the hypersensitive response, a particular type ofincompatible interaction occurring, without progressive diseasesymptoms. During compatible interactions on host plants, bacterialpopulations increase dramatically and progressive symptoms occur. Duringincompatible interactions, bacterial populations do not increase, andprogressive symptoms do not occur.

The hypersensitive response is a rapid, localized necrosis that isassociated with the active defense of plants against many pathogens(Kiraly, Z., “Defenses Triggered by the Invader: Hypersensitivity,”pages 201–224 in: Plant Disease: An Advanced Treatise, Vol. 5, J. G.Horsfall and E. B. Cowling, ed. Academic Press New York (1980); Klement,Z., “Hypersensitivity,” pages 149–177 in: Phytopathogenic Prokaryotes,Vol. 2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York(1982)). The hypersensitive response elicited by bacteria is readilyobserved as a tissue collapse if high concentrations (≧10⁷ cells/ml) ofa limited host-range pathogen like Pseudomonas syringae or Erwiniaamylovora are infiltrated into the leaves of nonhost plants (necrosisoccurs only in isolated plant cells at lower levels of inoculum)(Klement, Z., “Rapid Detection of Pathogenicity of PhytopathogenicPseudomonads,” Nature 199:299–300; Klement, et al., “HypersensitiveReaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf,”Phytopathology 54:474–477 (1963); Turner, et al., “The QuantitativeRelation Between Plant and Bacterial Cells Involved in theHypersensitive Reaction,” Phytopathology 64:885–890 (1974); Klement, Z.,“Hypersensitivity,” pages 149–177 in Phytopathogenic Prokaryotes, Vol.2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)).The capacities to elicit the hypersensitive response in a nonhost and bepathogenic in a host appear linked. As noted by Klement, Z.,“Hypersensitivity,” pages 149–177 in Phytopathogenic Prokaryotes, Vol.2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York, thesepathogens also cause physiologically similar, albeit delayed, necrosesin their interactions with compatible hosts. Furthermore, the ability toproduce the hypersensitive response or pathogenesis is dependent on acommon set of genes, denoted hrp (Lindgren, P. B., et al., “Gene Clusterof Pseudomonas syringae pv. ‘phaseolicola’ Controls Pathogenicity ofBean Plants and Hypersensitivity on Nonhost Plants,” J. Bacteriol.168:512–22 (1986); Willis, D. K., et al., “hrp Genes of PhytopathogenicBacteria,” Mol. Plant-Microbe Interact. 4:132–138 (1991)). Consequently,the hypersensitive response may hold clues to both the nature of plantdefense and the basis for bacterial pathogenicity.

The hrp genes are widespread in gram-negative plant pathogens, wherethey are clustered, conserved, and in some cases interchangeable(Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol.Plant-Microbe Interact. 4:132–138 (1991); Bonas, U., “hrp Genes ofPhytopathogenic Bacteria,” pages 79–98 in: Current Topics inMicrobiology and Immunology: Bacterial Pathogenesis of Plants andAnimals-Molecular and Cellular Mechanisms, J. L. Dangl, ed.Springer-Verlag, Berlin (1994)). Several hrp genes encode components ofa protein secretion pathway similar to one used by Yersinia, Shigella,and Salmonella spp. to secrete proteins essential in animal diseases(Van Gijsegem, et al., “Evolutionary Conservation of PathogenicityDeterminants Among Plant and Animal Pathogenic Bacteria,” TrendsMicrobiol. 1:175–180 (1993)). In E. amylovora, P. syringae, and P.solanacearum, hrp genes have been shown to control the production andsecretion of glycine-rich, protein elicitors of the hypersensitiveresponse (He, S. Y., et al. “Pseudomonas Syringae pv. SyringaeHarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicitsthe Hypersensitive Response in Plants,” Cell 73:1255–1266 (1993), Wei,Z.-H., et al., “HrpI of Erwinia amylovora Functions in Secretion ofHarpin and is a Member of a New Protein Family,” J. Bacteriol.175:7958–7967 (1993); Arlat, M. et al. “PopA 1, a Protein Which Inducesa Hypersensitive-like Response on Specific Petunia Genotypes, isSecreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J.13:543–553 (1994)).

The first of these proteins was discovered in E. amylovora Ea321, abacterium that causes fire blight of rosaceous plants, and wasdesignated harpin (Wei, Z.-M., et al, “Harpin, Elicitor of theHypersensitive Response Produced by the Plant Pathogen Erwiniaamylovora,” Science 257:85–88 (1992)). Mutations in the encoding hrpNgene revealed that harpin is required for E. amylovora to elicit ahypersensitive response in nonhost tobacco leaves and incite diseasesymptoms in highly susceptible pear fruit. The P. solanacearum GMI1000PopA1 protein has similar physical properties and also elicits thehypersensitive response in leaves of tobacco, which is not a host ofthat strain (Arlat, et al. “PopA1, a Protein Which Induces aHypersensitive-like Response on Specific Petunia Genotypes, is Secretedvia the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543–53(1994)). However, P. solanacearum popA mutants still elicit thehypersensitive response in tobacco and incite disease in tomato. Thus,the role of these glycine-rich hypersensitive response elicitors canvary widely among gram-negative plant pathogens.

Other plant pathogenic hypersensitive response elicitors have beenisolated, cloned, and sequenced. These include: Erwiniachrysanthemi(Bauer, et. al., “Erwinia chrysanthemi Harpin_(ECh):Soft-Rot Pathogenesis,” MPMI 8(4): 484–91(1995)); Erwinia carotovora(Cui, et. al., “The RsmA⁻ Mutants of Erwinia carotovora subsp.carotovora Strain Ecc71 Overexpress hrpN_(Ecc) and Elicit aHypersensitive Reaction-like Response in Tobacco Leaves,” MPMI 9(7):565–73 (1966)); Erwinia stewartii (Ahmad, et. al., “Harpin is notNecessary for the Pathogenicity of Erwinia stewartii on Maize,” 8thInt'l. Cong. Molec. Plant-Microb. Inter. Jul. 14–19, 1996 and Ahmad, et.al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartiion Maize,” Ann. Mtg. Am. Phytopath. Soc. Jul. 27–31, 1996); andPseudomonas syringae pv. syringae (WO 94/26782 to Cornell ResearchFoundation, Inc.).

The present invention is a further advance in the effort to identify,clone, and sequence hypersensitive response elicitor proteins orpolypeptides from plant pathogens.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated protein or polypeptidewhich elicits a hypersensitive response in plants as well as an isolatedDNA molecule which encodes the hypersensitive response eliciting proteinor polypeptide.

The hypersensitive response eliciting protein or polypeptide can be usedto impart disease resistance to plants, to enhance plant growth, and/orto control insects. This involves applying the hypersensitive responseelicitor protein or polypeptide in a non-infectious form to plants orplant seeds under conditions effective to impart disease resistance, toenhance plant growth, and/or to control insects on plants or plantsgrown from the plant seeds.

As an alternative to applying the hypersensitive response elicitorprotein or polypeptide to plants or plant seeds in order to impartdisease resistance, to enhance plant growth, and/or to control insectson plants, transgenic plants or plant seeds can be utilized. Whenutilizing transgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a hypersensitive responseelicitor protein or polypeptide and growing the plant under conditionseffective to impart disease resistance, to enhance plant growth, and/orto control insects in the plants or plants grown from the plant seeds.Alternatively, a transgenic plant seed transformed with the DNA moleculeencoding a hypersensitive response elicitor protein or polypeptide canbe provided and planted in soil. A plant is then propagated underconditions effective to impart disease resistance, to enhance plantgrowth, and/or to control insects on plants or plants grown from theplant seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and B show the molecular structure of the region of E.amylovora genome containing hrpW. FIG. 1A depicts cosmids pCPP430 andpCPP 450 that contain the regulatory and secretory region of the hrpcluster of E. amylovora. Arrow boxes on top of the cosmid clonesindicate the transcriptional units, where the names of the characterizedoperons are given above (Wei, et al., Science, 257:85–88 (1992); Zumoff,et al., The hrp Gene Cluster of Erwinia amylovora, eds. Hennecke, H. &Verma, D. P. S. (Kluwer Academic Publishers, Dordrecht, TheNetherlands), Vol. 1, pp. 53–60 (1991); Bogdanove, et al., J.Bacteriol., 178:1720–30 (1996); and Kim, et al., J. Bacteriol.,179:1690–97 (1997), which are hereby incorporated by reference). FIG. 1Bshows the location of hrpW which encodes a Gly-rich protein, andsubclones of pCPP1012 used in the study. Boxes and arrow boxes indicategenes or open reading frames; filled triangles are putativeHrpL-dependent promoters. Restriction sites: B, BamHI; E, EcoRI; H,HindIII, Ea, Eagl; Hp, Hpal.

FIG. 2 shows the expression of hrpW by a T7 RNA polymerase-directed geneexpression system. Lanes 1, E. coli DH5α(pGP1-2/pBC SK (−)); 2, E. coliDH5α(pGP1-2/pCPP1232). The arrow between 84 kD and 53 kD points to theband in lane 2 corresponding to the HrpW protein.

FIG. 3 shows the alignment of HrpW (SEQ ID NO: 2) with pectate lyases ofNectria haematococca, mating type VI (Fusarium solani f. sp. pisi)(P1A-Nh=SEQ ID NO: 4; P1B-Nh=SEQ ID NO: 5; P1C-Nh=SEQ ID NO: 6;P1D-Nh=SEQ ID NO: 7) and of Erwinia carotovora subsp. carotovora(Pel-3-Ec=SEQ ID NO: 8; PelB-Ec=SEQ ID NO: 9). The sequences werealigned by the PILEUP program (GCG software package, Version 7.3) withdefault parameters, and an alignment was manually edited using LINEUPprogram in the same package. Conserved residues are boxed, highlyconserved regions are underlined, and potential α-helices in HrpW areshaded. A consensus (SEQ ID NO: 10) within the Pel domain is shown belowthe alignment.

FIGS. 4A and B are immunoblots showing the hrp-dependent production andsecretion of HrpW in E. amylovora. Lanes in FIG. 4: 1, E. coliDH5α(pGP1–2/pCPP1232); 2, HrpN; 3, whole cell preparation (“CP”) ofEa321; 4, supernatant preparation (“SP”) of Ea321; 5, CP of Ea321-K49;6, SP of Ea321-K49; 7, CP of Ea321-G84; 8, SP of Ea321-G84. Lanes inFIG. 4B: 1, E. coli DH5α(pGP1–2/pCPP1232); 2, HrpN; 3, CP of Ea273; 4,SP of Ea273; 5, CP of Ea321-K49; 6, SP of Ea321-K49; 7, CP ofEa32,1-G73; 8, SP of Ea321-G73.

FIG. 5A shows a tobacco leaf showing residual hypersensitive response(“HR”) eliciting activity of hrpN mutants, and the HR induced by HrpNand HrpW. Panels: 1, E. coli DH5(pCPP430); 2, E. coli DH5(pCPP430-T5);3, E. coli MC4100(pCPP450); 4, E. coli MC4100(pCPP450-T5); 5, 5 mM KPO₄buffer (pH 6.5); 6, E. amylovora Ea321; 7, E. amylovora Ea321-T5; 8,HrpN CFEP (contains 0.5 mg/ml of HrpN); 9, HrpW preparation (0.5 mg/ml)eluted from the gel containing proteins from E. coliDH5α(pGP1–2/pCPP1232); 10, preparation made from E. coli DH5α(pGP1–2/pBCSK (−)) in the same manner as 9. The picture was taken 3 days afterinfiltration.

FIG. 5B shows suppression of the HrpW-induced HR by inhibitors of plantmetabolism. Panels: 1, 5 mM KPO₄ buffer (pH 6.5); 2, HrpW CFEP; 3, HrpNCFEP+cycloheximide; 4, HrpW CFEP+LaCl₃; 5, HrpW CFEP+Na₃VO₄; 6, HrpNCFEP; 7, HrpN CFEP+cycloheximide; 8, HrpN CFEP+Na₃VO₄; 9, PelE in 10 mMTris-HCI (pH 7.8); 10, PelE+cycloheximide; 11, PelE+Na₃VO₄. CFEPscontain 0.1 mg/ml of HrpW of HrpN. The picture of the tobacco leaf wastaken 36 hours after infiltration.

FIG. 6 shows a Southern blot which indicates that hrpW of E. amylovoraEa321 is present in other bacteria. Genomic DNA of strains was probedwith a 1.4-kb Hpal fragment that contains Ea321 hrpW. Lanes: 1, Ea321;2, Ea266; 3, Ea273; 4, Ea246; 5, Ea510; 6, Ea528; 7, Ea574; 8, Ea546; 9,Ea557; 10, Ea562; 11. Ea587; 12, E. carotovora subsp. carotovoraATCC15713; 13, E. mallotivora 1818; 14, E. salicis 1822; 15, pCPP2157 ofE. chrysanthemi EC16.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated DNA molecule having anucleotide sequence of SEQ. ID. No. 1 as follows:

ATGTCAATTC TTACGCTTAA CAACAATACC TCGTCCTCGC CGGGTCTGTT CCAGTCCGGG   60GGGGACAACG GGCTTGGTGG TCATAATGCA AATTCTGCGT TGGGGCAACA ACCCATCGAT  120CGGCAAACCA TTGAGCAAAT GGCTCAATTA TTGGCGGAAC TGTTAAAGTC ACTGCTATCG  180CCACAATCAG GTAATGCGGC AACCGGAGCC GGTGGCAATG ACCAGACTAC AGGAGTTGGT  240AACGCTGGCG GCCTGAACGG ACGAAAAGGC ACAGCAGGAA CCACTCCGCA GTCTGACAGT  300CAGAACATGC TGAGTGAGAT GGGCAACAAC GGGCTGGATC AGGCCATCAC GCCCGATGGC  360CAGGGCGGCG GGCAGATCGG CGATAATCCT TTACTGAAAG CCATGCTGAA GCTTATTGCA  420CGCATGATGG ACGGCCAAAG CGATCAGTTT GGCCAACCTG GTACGGGCAA CAACAGTGCC  480TCTTCCGGTA CTTCTTCATC TGGCGGTTCC CCTTTTAACG ATCTATCAGG GGGGAAGGCC  540CCTTCCGGCA ACTCCCCTTC CGGCAACTAC TCTCCCGTCA GTACCTTCTC ACCCCCATCC  600ACGCCAACGT CCCCTACCTC ACCGCTTGAT TTCCCTTCTT CTCCCACCAA AGCAGCCGGG  660GGCAGCACGC CGGTAACCGA TCATCCTGAC CCTGTTGGTA GCGCGGGCAT CGGGGCCGGA  720AATTCGGTGG CCTTCACCAG CGCCGGCGCT AATCAGACGG TGCTGCATGA CACCATTACC  780GTGAAAGCGG GTCAGGTGTT TGATGGCAAA GGACAAACCT TCACCGCCGG TTCAGAATTA  840GGCGATGGCG GCCAGTCTGA AAACCAGAAA CCGCTGTTTA TACTGGAAGA CGGTGCCAGC  900CTGAAAAACG TCACCATGGG CGACGACGGG GCGGATGGTA TTCATCTTTA CGGTGATGCC  960AAAATAGACA ATCTGCACGT CACCAACGTG GGTGAGGACG CGATTACCGT TAAGCCAAAC 1020AGCGCGGGCA AAAAATCCCA CGTTGAAATC ACTAACAGTT CCTTCGAGCA CGCCTCTGAC 1080AAGATCCTGC AGCTGAATGC CGATACTAAC CTGAGCGTTG ACAACGTGAA GGCCAAAGAC 1140TTTGGTACTT TTGTACGCAC TAACGGCGGT CAACAGGGTA ACTGGGATCT GAATCTGAGC 1200CATATCAGCG CAGAAGACGG TAAGTTCTCG TTCGTTAAAA GCGATAGCGA GGGGCTAAAC 1260GTCAATACCA GTGATATCTC ACTGGGTGAT GTTGAAAACC ACTACAAAGT GCCGATGTCC 1320GCCAACCTGA AGGTGGCTGA ATGA 1344See GenBank Accession No. U94513. The isolated DNA molecule of thepresent invention encodes a hypersensitive response elicitor protein orpolypeptide having an amino acid sequence of SEQ. ID. No. 2 as follows:

Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu1               5                   10                  15 Phe Gln SerGly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser            20                  25                  30 Ala Leu Gly GlnGln Pro Ile Asp Arg Gln Thr Ile Glu Gln Met Ala        35                  40                  45 Gln Leu Leu Ala GluLeu Leu Lys Ser Leu Leu Ser Pro Gln Ser Gly    50                  55                  60 Asn Ala Ala Thr Gly AlaGly Gly Asn Asp Gln Thr Thr Gly Val Gly65                  70                  75                  80 Asn AlaGly Gly Leu Asn Gly Arg Lys Gly Thr Ala Gly Thr Thr Pro                85                  90                  95 Gln Ser AspSer Gln Asn Met Leu Ser Glu Met Gly Asn Asn Gly Leu            100                 105                 110 Asp Gln Ala IleThr Pro Asp Gly Gln Gly Gly Gly Gln Ile Gly Asp        115                 120                 125 Asn Pro Leu Leu LysAla Met Leu Lys Leu Ile Ala Arg Met Met Asp    130                 135                 140 Gly Gln Ser Asp Gln PheGly Gln Pro Gly Thr Gly Asn Asn Ser Ala145                 150                 155                 160 Ser SerGly Thr Ser Ser Ser Gly Gly Ser Pro Phe Asn Asp Leu Ser                165                 170                 175 Gly Gly LysAla Pro Ser Gly Asn Ser Pro Ser Gly Asn Tyr Ser Pro            180                 185                 190 Val Ser Thr PheSer Pro Pro Ser Thr Pro Thr Ser Pro Thr Ser Pro        195                 200                 205 Leu Asp Phe Pro SerSer Pro Thr Lys Ala Ala Gly Gly Ser Thr Pro    210                 215                 220 Val Thr Asp His Pro AspPro Val Gly Ser Ala Gly Ile Gly Ala Gly225                 230                 235                 240 Asn SerVal Ala Phe Thr Ser Ala Gly Ala Asn Gln Thr Val Leu His                245                 250                 255 Asp Thr IleThr Val Lys Ala Gly Gln Val Phe Asp Gly Lys Gly Gln            260                 265                 270 Thr Phe Thr AlaGly Ser Glu Leu Gly Asp Gly Gly Gln Ser Glu Asn        275                 280                 285 Gln Lys Pro Leu PheIle Leu Glu Asp Gly Ala Ser Leu Lys Asn Val    290                 295                 300 Thr Met Gly Asp Asp GlyAla Asp Gly Ile His Leu Tyr Gly Asp Ala305                 310                 315                 320 Lys IleAsp Asn Leu His Val Thr Asn Val Gly Glu Asp Ala Ile Thr                325                 330                 335 Val Lys ProAsn Ser Ala Gly Lys Lys Ser His Val Glu Ile Thr Asn            340                 345                 350 Ser Ser Phe GluHis Ala Ser Asp Lys Ile Leu Gln Leu Asn Ala Asp        355                 360                 365 Thr Asn Leu Ser ValAsp Asn Val Lys Ala Lys Asp Phe Gly Thr Phe    370                 375                 380 Val Arg Thr Asn Gly GlyGln Gln Gly Asn Trp Asp Leu Asn Leu Ser385                 390                 395                 400 His IleSer Ala Glu Asp Gly Lys Phe Ser Phe Val Lys Ser Asp Ser                405                 410                 415 Glu Gly LeuAsn Val Asn Thr Ser Asp Ile Ser Leu Gly Asp Val Glu            420                 425                 430 Asn His Tyr LysVal Pro Met Ser Ala Asn Leu Lys Val Ala Glu        435                 440                 445

This protein or polypeptide is acidic, rich in glycine and serine, andlacks cysteine. It is also heat stable, protease sensitive, andsuppressed by inhibitors of plant metabolism. The protein or polypeptideof the present invention has a predicted molecular size of ca. 45 kDa.

Fragments of the above hypersensitive response elicitor polypeptide orprotein are encompassed by the the present invention.

Suitable fragments can be produced by several means. In the first,subclones of the gene encoding the elicitor protein of the presentinvention are produced by conventional molecular genetic manipulation bysubcloning gene fragments. The subclones then are expressed in vitro orin vivo in bacterial cells to yield a smaller protein or peptide thatcan be tested for elicitor activity according to the procedure describedbelow.

As an alternative, fragments of an elicitor protein can be produced bydigestion of a full-length elicitor protein with proteolytic enzymeslike chymotrypsin or Staphylococcus proteinase A, or trypsin. Differentproteolytic enzymes are likely to cleave elicitor proteins at differentsites based on the amino acid sequence of the elicitor protein. Some ofthe fragments that result from proteolysis may be active elicitors ofresistance.

In another approach, based on knowledge of the primary structure of theprotein, fragments of the elicitor protein gene may be synthesized byusing the PCR technique together with specific sets of primers chosen torepresent particular portions of the protein. These then would be clonedinto an appropriate vector for increased expression of a truncatedpeptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for theelicitor being produced. Alternatively, subjecting a full lengthelicitor to high temperatures and pressures will produce fragments.These fragments can then be separated by conventional procedures (e.g.,chromatography, SDS-PAGE).

Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theproperties, secondary structure and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Suitable DNA molecules are those that hybridize to a DNA moleculecomprising a nucleotide sequence of SEQ. ID. No. 1 under stringentconditions. An example of suitable stringency conditions is whenhybridization is carried out at 65° C. for 20 hours in a mediumcontaining 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodiumdodecyl sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovineserum albumin, 50 μm g/ml E. coli DNA. However, any DNA moleculeshybridizing to a DNA molecule comprising the nucleotide sequence of SEQ.ID. No. 1 under such stringent conditions must not be identical to thenucleic acids encoding the hypersensitive response elicitor proteins orpolypeptides of E. amylovora (as disclosed by Wei, Z.-M., et al,“Harpin, Elicitor of the Hypersensitive Response Produced by the PlantPathogen Erwinia amylovora,” Science 257:85–88 (1992), which is herebyincorporated by reference), Erwinia chrysanthemi (as disclosed by Bauer,et. al., “Erwinia chrysanthemi Harpin_(ECh): Soft-Rot Pathogenesis,”MPMI 8(4): 484–91 (1995), which is hereby incorporated by reference),Erwinia carotovora (as disclosed by Cui, et. al., “The RsmA⁻ Mutants ofErwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrpN_(ECC)and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,”MPMI 9(7): 565–73 (1966), which is hereby incorporated by reference),Erwinia stewartii (as disclosed by Ahmad, et. al., “Harpin is notNecessary for the Pathogenicity of Erwinia stewartii on Maize,” 8thInt'l. Cong. Molec. Plant-Microb. Inter. Jul. 14–19, 1996 and Ahmad, et.al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartiion Maize,” Ann. Mtg. Am. Phytopath. Soc. Jul. 27–31, 1996), which arehereby incorporated by reference), and Pseudomonas syringae pv. syringae(WO 94/26782 to Cornell Research Foundation, Inc., which is herebyincorporated by reference).

The protein or polypeptide of the present invention is preferablyproduced in purified form (preferably at least about 80%, morepreferably 90%, pure) by conventional techniques. Typically, the proteinor polypeptide of the present invention is secreted into the growthmedium of recombinant host cells. Alternatively, the protein orpolypeptide of the present invention is produced but not secreted intogrowth medium. In such cases, to isolate the protein, the host cell(e.g., E. coli) carrying a recombinant plasmid is propagated, lysed bysonication, heat, differential pressure, or chemical treatment, and thehomogenate is centrifuged to remove bacterial debris. The supernatant isthen subjected to sequential ammonium sulfate precipitation. Thefraction containing the polypeptide or protein of the present inventionis subjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the proteins. If necessary, theprotein fraction may be further purified by HPLC.

The DNA molecule encoding the hypersensitive response elicitorpolypeptide or protein can be incorporated in cells using conventionalrecombinant DNA technology. Generally, this involves inserting the DNAmolecule into an expression system to which the DNA molecule isheterologous (i.e. not normally present). The heterologous DNA moleculeis inserted into the expression system or vector in proper senseorientation and correct reading frame. The vector contains the necessaryelements for the transcription and translation of the insertedprotein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference, describes the production of expression systems in the formof recombinant plasmids using restriction enzyme cleavage and ligationwith DNA ligase. These recombinant plasmids are then introduced by meansof transformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinavirus. Recombinant viruses can be generated by transfection of plasmidsinto cells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/−or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated byreference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al.,“Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” GeneExpression Technology vol. 185 (1990), which is hereby incorporated byreference), and any derivatives thereof. Recombinant molecules can beintroduced into cells via transformation, particularly transduction,conjugation, mobilization, or electroporation. The DNA sequences arecloned into the vector using standard cloning procedures in the art, asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which ishereby incorporated by reference.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria. Theexpression elements of these vectors vary in their strength andspecificities. Depending upon the host-vector system utilized, any oneof a number of suitable transcription and translation elements can beused.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (mRNA)translation).

Transcription of DNA is dependent upon the presence of a promotor whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eucaryotic promotorsdiffer from those of procaryotic promotors. Furthermore, eucaryoticpromotors and accompanying genetic signals may not be recognized in ormay not function in a procaryotic system, and, further, procaryoticpromoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eucaryotes.Efficient translation of mRNA in procaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference.

Promotors vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promotors in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promotors maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promotor, trppromotor, recA promotor, ribosomal RNA promotor, the P_(R) and P_(L)promotors of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promotor or other E. coli promotors produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promotor unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promotor, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7–9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the N gene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

Once the isolated DNA molecule encoding the hypersensitive responseelicitor polypeptide or protein has been cloned into an expressionsystem, it is ready to be incorporated into a host cell. Suchincorporation can be carried out by the various forms of transformationnoted above, depending upon the vector/host cell system. Suitable hostcells include, but are not limited to, bacteria, virus, yeast, mammaliancells, insect, plant, and the like.

The present invention further relates to methods of imparting diseaseresistance to plants, enhancing plant growth, and/or effecting insectcontrol for plants. These methods involve applying a hypersensitiveresponse elicitor polypeptide or protein in a non-infectious form to allor part of a plant or a plant seed under conditions where thepolypeptide or protein contacts all or part of the cells of the plant orplant seed. Alternatively, the hypersensitive response elicitor proteinor polypeptide can be applied to plants such that seeds recovered fromsuch plants themselves are able to impart disease resistance in plants,to enhance plant growth, and/or to effect insect control.

As an alternative to applying a hypersensitive response elicitorpolypeptide or protein to plants or plant seeds in order to impartdisease resistance in plants, to effect plant growth, and/or to controlinsects on the plants or plants grown from the seeds, transgenic plantsor plant seeds can be utilized. When utilizing transgenic plants, thisinvolves providing a transgenic plant transformed with a DNA moleculeencoding a hypersensitive response elicitor polypeptide or protein andgrowing the plant under conditions effective to permit that DNA moleculeto impart disease resistance to plants, to enhance plant growth, and/orto control insects. Alternatively, a transgenic plant seed transformedwith a DNA molecule encoding a hypersensitive response elicitorpolypeptide or protein can be provided and planted in soil. A plant isthen propagated from the planted seed under conditions effective topermit that DNA molecule to impart disease resistance to plants, toenhance plant growth, and/or to control insects.

The embodiment of the present invention where the hypersensitiveresponse elicitor polypeptide or protein is applied to the plant orplant seed can be carried out in a number of ways, including: 1)application of an isolated elicitor polypeptide or protein; 2)application of bacteria which do not cause disease and are transformedwith genes encoding a hypersensitive response elicitor polypeptide orprotein; and 3) application of bacteria which cause disease in someplant species (but not in those to which they are applied) and naturallycontain a gene encoding the hypersensitive response elicitor polypeptideor protein.

In one embodiment of the present invention, the hypersensitive responseelicitor polypeptide or protein of the present invention can be isolatedfrom Erwinia amylovora as described in Examples infra. Preferably,however, the isolated hypersensitive response elicitor polypeptide orprotein of the present invention is produced recombinantly and purifiedas described supra.

In other embodiments of the present invention, the hypersensitiveresponse elicitor polypeptide or protein of the present invention can beapplied to plants or plant seeds by applying bacteria containing genesencoding the hypersensitive response elicitor polypeptide or protein.Such bacteria must be capable of secreting or exporting the polypeptideor protein so that the elicitor can contact plant or plant seeds cells.In these embodiments, the hypersensitive response elicitor polypeptideor protein is produced by the bacteria in planta or on seeds or justprior to introduction of the bacteria to the plants or plant seeds.

In one embodiment of the bacterial application mode of the presentinvention, the bacteria do not cause the disease and have beentransformed (e.g., recombinantly) with genes encoding a hypersensitiveresponse elicitor polypeptide or protein. For example, E. coli, whichdoes not elicit a hypersensitive response in plants, can be transformedwith genes encoding a hypersensitive response elicitor polypeptide orprotein and then applied to plants. Bacterial species other than E. colican also be used in this embodiment of the present invention.

In another embodiment of the bacterial application mode of the presentinvention, the bacteria do cause disease and naturally contain a geneencoding a hypersensitive response elicitor polypeptide or protein.Examples of such bacteria are noted above. However, in this embodiment,these bacteria are applied to plants or their seeds which are notsusceptible to the disease carried by the bacteria. For example, Erwiniaamylovora causes disease in apple or pear but not in tomato. However,such bacteria will elicit a hypersensitive response in tomato.Accordingly, in accordance with this embodiment of the presentinvention, Erwinia amylovora can be applied to tomato plants or seeds toimpart disease resistance, enhance growth, or control insects withoutcausing disease in that species.

The method of the present invention can be utilized to treat a widevariety of plants or their seeds to impart disease resistance, enhancegrowth, and/or control insects. Suitable plants include dicots andmonocots. More particularly, useful crop plants can include: alfalfa,rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweetpotato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout,beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach,onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin,zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape,raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.Examples of suitable ornamental plants are: Arabidopsis thaliana,Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation,and zinnia.

With regard to the use of the hypersensitive response elicitor proteinor polypeptide of the present invention in imparting disease resistance,absolute immunity against infection may not be conferred, but theseverity of the disease is reduced and symptom development is delayed.Lesion number, lesion size, and extent of sporulation of fungalpathogens are all decreased. This method of imparting disease resistancehas the potential for treating previously untreatable diseases, treatingdiseases systemically which might not be treated separately due to cost,and avoiding the use of infectious agents or environmentally harmfulmaterials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi. Resistance,inter alia, to the following viruses can be achieved by the method ofthe present invention: Tobacco mosaic virus and Tomato mosaic virus.Resistance, inter alia, to the following bacteria can also be impartedto plants in accordance with present invention: Pseudomonas solancearum,Pseudomonas syringae pv. tabaci, and Xanthamonas campestris pv.pelargonii. Plants can be made resistant, inter alia, to the followingfungi by use of the method of the present invention: Fusarium oxysporumand Phytophthora infestans.

With regard to the use of the hypersensitive response elicitor proteinor polypeptide of the present invention to enhance plant growth, variousforms of plant growth enhancement or promotion can be achieved. This canoccur as early as when plant growth begins from seeds or later in thelife of a plant. For example, plant growth according to the presentinvention encompasses greater yield, increased quantity of seedsproduced, increased percentage of seeds germinated, increased plantsize, greater biomass, more and bigger fruit, earlier fruit coloration,and earlier fruit and plant maturation. As a result, the presentinvention provides significant economic benefit to growers. For example,early germination and early maturation permit crops to be grown in areaswhere short growing seasons would otherwise preclude their growth inthat locale. Increased percentage of seed germination results inimproved crop stands and more efficient seed use. Greater yield,increased size, and enhanced biomass production allow greater revenuegeneration from a given plot of land.

Another aspect of the present invention is directed to effecting anyform of insect control for plants. For example, insect control accordingto the present invention encompasses preventing insects from contactingplants to which the hypersensitive response elicitor has been applied,preventing direct insect damage to plants by feeding injury, causinginsects to depart from such plants, killing insects proximate to suchplants, interfering with insect larval feeding on such plants,preventing insects from colonizing host plants, preventing colonizinginsects from releasing phytotoxins, etc. The present invention alsoprevents subsequent disease damage to plants resulting from insectinfection.

The present invention is effective against a wide variety of insects.European corn borer is a major pest of corn (dent and sweet corn) butalso feeds on over 200 plant species including green, wax, and limabeans and edible soybeans, peppers, potato, and tomato plus many weedspecies. Additional insect larval feeding pests which damage a widevariety of vegetable crops include the following: beet armyworm, cabbagelooper, corn ear worm, fall armyworm, diamondback moth, cabbage rootmaggot, onion maggot, seed corn maggot, pickleworm (melonworm), peppermaggot, and tomato pinworm. Collectively, this group of insect pestsrepresents the most economically important group of pests for vegetableproduction worldwide.

The method of the present invention involving application of thehypersensitive response elicitor polypeptide or protein can be carriedout through a variety of procedures when all or part of the plant istreated, including leaves, stems, roots, propagules (e.g., cuttings),etc. This may (but need not) involve infiltration of the hypersensitiveresponse elicitor polypeptide or protein into the plant. Suitableapplication methods include high or low pressure spraying, injection,and leaf abrasion proximate to when elicitor application takes place.When treating plant seeds, in accordance with the application embodimentof the present invention, the hypersensitive response elicitor proteinor polypeptide can be applied by low or high pressure spraying, coating,immersion, or injection. Other suitable application procedures can beenvisioned by those skilled in the art provided they are able to effectcontact of the hypersensitive response elicitor polypeptide or proteinwith cells of the plant or plant seed. Once treated with thehypersensitive response elicitor of the present invention, the seeds canbe planted in natural or artificial soil and cultivated usingconventional procedures to produce plants. After plants have beenpropagated from seeds treated in accordance with the present invention,the plants may be treated with one or more applications of thehypersensitive response elicitor protein or polypeptide to impartdisease resistance to plants, to enhance plant growth, and/or to controlinsects on the plants.

The hypersensitive response elicitor polypeptide or protein can beapplied to plants or plant seeds in accordance with the presentinvention alone or in a mixture with other materials. Alternatively, thehypersensitive response elicitor polypeptide or protein can be appliedseparately to plants with other materials being applied at differenttimes.

A composition suitable for treating plants or plant seeds in accordancewith the application embodiment of the present invention contains ahypersensitive response elicitor polypeptide or protein in a carrier.Suitable carriers include water, aqueous solutions, slurries, or drypowders. In this embodiment, the composition contains greater than 500nM hypersensitive response elicitor polypeptide or protein.

Although not required, this composition may contain additional additivesincluding fertilizer, insecticide, fungicide, nematacide, and mixturesthereof. Suitable fertilizers include (NH₄)₂NO₃. An example of asuitable insecticide is Malathion. Useful fungicides include Captan.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate the process of the present invention. In addition, thehypersensitive response elicitor polypeptide or protein can be appliedto plant seeds with other conventional seed formulation and treatmentmaterials, including clays and polysaccharides.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a hypersensitive responseelicitor polypeptide or protein need not be applied optically to theplants or seeds. Instead, transgenic plants transformed with a DNAmolecule encoding a hypersensitive response elicitor polypeptide orprotein are produced according to procedures well known in the art.

The vector described above can be microinjected directly into plantcells by use of micropipettes to transfer mechanically the recombinantDNA. Crossway, Mol. Gen. Genetics, 202:179–85 (1985), which is herebyincorporated by reference. The genetic material may also be transferredinto the plant cell using polyethylene glycol. Krens, et al., Nature,296:72–74 (1982), which is hereby incorporated by reference.

Another approach to transforming plant cells with a gene which impartsresistance to pathogens is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859–63 (1982), which is hereby incorporated by reference.

The DNA molecule may also be introduced into the plant cells byelectroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference. In this technique,plant protoplasts are electroporated in the presence of plasmidscontaining the expression cassette. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the plasmids. Electroporated plant protoplasts reform the cell wall,divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25–28° C.

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. Its species are responsible for crown gall (A.tumefaciens) and hairy root disease (A. rhizogenes). The plant cells incrown gall tumors and hairy roots are induced to produce amino acidderivatives known as opines, which are catabolized only by the bacteria.The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome. J. Schell, Science, 237:1176–83 (1987), which is herebyincorporated by reference.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III(1986), which are hereby incorporated by reference.

It is known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to, all major species ofsugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure with thepresence of the gene encoding the hypersensitive response elicitorresulting in disease resistance, enhanced plant growth, and/or controlof insects on the plant. Alternatively, transgenic seeds are recoveredfrom the transgenic plants. These seeds can then be planted in the soiland cultivated using conventional procedures to produce transgenicplants. The transgenic plants are propagated from the planted transgenicseeds under conditions effective to impart disease resistance to plants,to enhance plant growth, and/or to control insects. While not wishing tobe bound by theory, such disease resistance, growth enhancement, and/orinsect control may be RNA mediated or may result from expression of theelicitor polypeptide or protein.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used to treat the plants and seeds to which ahypersensitive response elicitor polypeptide or protein is applied.These other materials, including hypersensitive response elicitors, canbe applied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the hypersensitive response elicitor to impart diseaseresistance, enhance growth, and/or control insects. Such plants may alsobe treated with conventional plant treatment agents (e.g., insecticides,fertilizers, etc.).

EXAMPLES Example 1 Bacterial Strains and Plasmids

E. amylovora Ea321 and Ea273 are wild-type strains that infect pomaceousplants (Beer et al., The hrp Gene Cluster of Erwinia amylovora, eds.Hennecke, H. & Verma, D. P. S. (Kluwer Academic Publishers, Dordrecht,The Netherlands), Vol. 1, pp. 53–50 (1991), which is hereby incorporatedby reference). Escherichia coli DH5α was used routinely as the host ofplasmids. pCPP1012 is a subclone of pCPP430, and pCPP1152, pCPP1218,pCPP1219 and pCPP1220 were constructed by cloning restriction fragmentsof pCPP1012 into pBluescript KS (+) (Stratagene, La Jolla, Calif.) (FIG.1B). pCPP1227 was cloned from pCPP 1220 into the same vector.

Example 2 Molecular Biological Techniques and Sequence Analysis

General molecular procedures were performed using standard techniques asdescribed (Sambrook, et al., Molecular Cloning: A Laboratory Manual,(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) (1989), whichis hereby incorporated by reference). Sequencing was done on an ABI 373Aautomated DNA sequencer at the Cornell University Biotechnology ProgramDNA Sequencing Facility. For DNA and protein sequence analyses, programsin the GCG software package, Version 7.3 (Genetics Computer Group, Inc.,Madison, Wis.) and DNASTAR (DNASTAR, Inc., Madison, Wis.) were used.

Example 3 Expression of hrpW in E. coli

The 1.4-kb Hpal fragment of pCPP1227 that contains hrpW was subclonedinto pBC SK (−) (Stratagene, La Jolla, Calif.) such that hrpW is underthe control of T7Φ10 promoter. The resulting plasmid, pCPP1232 (FIG.1B), was introduced into E. coli DH5α((pGP1–2) (Tabor, et al., Proc.Natl. Acad. Sci. USA, 82:1074–78 (1985), which is hereby incorporated byreference). Cells were incubated at 42° C. to induce the expression ofthe T7 RNA polymerase gene, and newly synthesized proteins wereradiolabelled with ³⁵S-Met as described (Tabor, et al., Proc. Natl.Acad. Sci. USA, 82:1074–78 (1985), which is hereby incorporated byreference). Resulting samples were resuspended in a crackling buffer andheated to 95° C. for 3 min before SDS-polyacrylamide gel electrophoresis(SDS-PAGE) in a 10% gel.

Example 4 Purification of HrpW

HrpW, produced by heat-shock treatment of E. coli DH5α(pGP1–2, pCPP1232)at 42° C., was purified by cutting out the area of the gel containingHrpW, eluting the protein with ELUTRAP (Schleicher & Schuell, Inc.,Keene, N.H.), and desalting the HrpW-containing solution usingCentriprep-30 (Amicon, Inc., Beverly, Mass.) and 5 mM potassiumphosphate (KPO₄) buffer (pH 6.5). Alternatively, heat-induced and10-fold concentrated E. coli DH5α(pGP1–2, pCPP1232) cells were sonicatedin the presence of 1 mM phenylmethlsulfonyl fluoride (PMSF), put in aboiling water bath for 10 min, and centrifuged at 17,500 g for 10 min.The supernatant was desalted resulting in a “cell-free elicitorpreparation (CFEP)” of HrpW. The CFEP of HrpW was prepared in the samemanner from an HrpN overproducer, E. coli DH5α(pCPP2139).

Example 5 Immunodetection of HrpW

Polyclonal antibodies against HrpW were raised at the College ofVeterinary Medicine, Cornell University, by injecting ca. 100 μg of HrpWinto a rabbit three times at 2–3 wk intervals. The antiserum wascollected 2 wk after the final injection and cross-absorbed withheat-treated lysate of E. coli DH5α(pGP1–2, pBC SK (−)).

E. amylovora Ea321Rp (a rifampicin-resistant derivative of Ea321),Ea321-K49 (hrpL::Tn 10-miniKm) (Wei, et al., J. Bacteriol.,177:6201–10(1995), which is hereby incorporated by reference), Ea321-G84(hrcC::Tn5-gusA1) (Kim et al., J. Bacteriol., 179:1690–97 (1997), whichis hereby incorporated by reference), Ea273Rp, Ea273-K49, and Ea273-G73(hrcV::Tn5-gusA1) were grown overnight in Terrific broth, transferred toa hrp minimal medium (Huynh, et al., Science 345:1374–77 (1989), whichis hereby incorporated by reference) at 1×10⁸ cfu/ml, and incubated at20° C. until the bacteria grew to 1×10⁹ cfu/ml. Cultures werecentrifuged at 17,500 g and the pellet was resuspended in a loadingbuffer. The supernatant was passed through a membrane filter (0.2 μmpore size; Whatman Inc., Fairfield, N.J.) after adding 1 mM PMSF, andconcentrated 100-fold using Centricon-10 and Microcon-10 (Amicon, Inc.,Beverly, Mass.) at 4° C. Both the cell and supernatant fractions werethen subjected to SDS-PAGE in a 10% gel.

Proteins in the gel were transferred to Immobilon-P (Millipore Co.,Bedford, Mass.) and western analysis was performed using a system(Sigma, St. Louis, Mo.) composed of Biotin-conjugated anti-rabbit IgG,ExtrAvidin, and BCIP/NBT tablets for strains of Ea273 and E. coli, andusing the Western-Light Plus system (Tropix, Inc., Bedford, Mass.) forstrains of Ea321.

Example 6 Generation of an N-terminal Fragment of HrpW

pCPP1232 was digested with BamHI and BstEII and the ends of the 4.1-kbfragment were blunted using the Klenow fragment and self-ligated. Theresulting plasmid, pCPP1254, which encodes the N-terminal 226 aminoacids of HrpW and IIe-His residues derived from the vector sequence, wascloned in E. coli DH5α, and then transferred to E. coli DH5α(pGP1–2),generating E. coli DH5α(pGP1–2, pCPP1254).

Example 7 Plant Assays

Elicitation of the HR was tested by infiltrating protein or bacterialpreparations into the intercellular space of leaves of tobacco(Nicotiana tabacum L. ‘xanthi’) and other plants (Kim, et al., J.Bacteriol., 179:1690–97 (1997), which is hereby incorporated byreference). Cells were grown either in Luria broth (E. coli DH5α andMC4100) or a hrp minimal medium (E. amylovora Ea321 and Ea321–T5)(Huynh, et al., Science 345:1374–77 (1989), which is hereby incorporatedby reference) to 5×10⁸ cfu/ml, and resuspended in 5 mM KPO₄ buffer (pH6.5) to 2×10⁸ cfu/ml (E. coli strains) or 5×10⁸ cfu/ml (E. amylovorastrains). Inhibitors of plant metabolism used included cycloheximide at100 μM, LaCl₃ at 1 mM, and Na₃VO₄ at 50 μM.

Example 8 Southern Blotting

Genomic DNA was digested with EcoRI, electrophoresed on a 0.7% agarosegel, transferred to an Immobilon-N membrane (Millipore Co., Bedford,Mass.), and hybridized with the ³²P-Labelled 1.4 kb-Hpal fragment ofpCPP1227 at 65° C. for 24 hr in a hybridization solution of 6×SSC, 5×Denhardt's reagent, 0.5% SDS, and 100 μg/ml denatured fragmented salmonsperm DNA (Sambrook et al., Molecular Cloning: A Laboratory Manual,(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) (1989) at9.31–9.57. The membrane was washed twice with a solution of 2×SSC and1.0% SDS at 65° C., and washed with 0.1×SSC until no radioactivity isdetected in the wash solution. For low stringency hybridizations, themembrane was incubated at 50° C. and washed with 2×SSC at 45° C.

Example 9 Pectic Enzyme Assay of HrpW

Heat-induced E. coli DH5α(pGP1–2, pCPP1232) were pelleted, resuspendedin one-tenth volume of 5 mM KPO₄ buffer (pH 6.5) or 10 mM Tris-HCl (pH8.5), sonicated on ice, centrifuged, and PL activity of the supernatantwas tested. Also, 50-fold concentrated Ea321 culture supernatant wasincluded in the test. Dilute PelE of Erwinia Chrysanthemi EC16 in 10 mMTris-HCl (pH 7.8) was used as a control.

Ten microliters of each preparation was spotted in YC agar plates (Keen,et al., J. Bacteriol., 159:825–31 (1984), which is hereby incorporatedby reference) containing either 0.7% polygalaturonic acid (Sigma, St.Louis, Mo.) or 0.7% pectin (88% methoxylated; Sigma, St. Louis, Mo.) atpH 6.5, 8.0 or 9.5, and in pectin semi-solid agar plates (Starr, et al.,J. Clin. Microbiol., 6:379–86 (1977), which is hereby incorporated byreference) containing 3% pectin (88% methoxylated) at pH 6.5, 8.0 or9.5. The plates were incubated at 37° C. for 24 hr, flooded with 1 MCaCl₂, and examined for the presence of halo. Viscometric analysis(Bateman, D. F., Phytopathology, 53:197–204 (1963), which is herebyincorporated by reference) and a modified thioarbituric acid procedure(Sherwood, R. T., Phytopathology, 56:279–86 (1966), which is herebyincorporated by reference) was done using 1% polygalaturonic acid or 1%pectin (68% methoxylated) in 2.5 mM CaCl₂ and 100 mM Tris-HCl, pH 9.5 assubstrates. Methods supposedly more sensitive than those above,including an isoelectrofocusing gel procedure and spectrophotometry alsowere tried. Ten microliters of samples was applied onto an overlay gel(Collmer, et al., J. Bacteriol., 161:913–20 (1985), which is herebyincorporated by reference) containing 0.2% pectin (88% methoxylated), 1%agarose, 1.5 mM CaCl₂, and 50 mM Tris-HCl (pH 8.8), and wrapped withplastic film. The gel was incubated at 28° C. for 24 hr, flooded with 1%hexadecyltrimethylammonium bromide, and inspected for clearing. To assayPL activity through generation of double bonds, 1.9 ml of a solution of0.1% pectin (68% methoxylated), 5 mM CaCl₂, and 100 mM Tris-HCI at pH5.5 or 9.5 was mixed with 100μl of samples and absorbance changes at 232nm was recorded for 30 min as described (Alfano, et al., J. Bacteriol.,177:4553–56 (1995), which is hereby incorporated by reference).

Example 10 Identification of a Gene Encoding a Glycine-Rich Protein

hrpN mutants pCPP430-T5 and pCPP450-T5 in E. coli exhibited residualHR-eliciting activity (FIG. 5A, panels 2 and 4), suggesting theexistence of another HR elicitor in the clones. The DNA downstream ofhrpN, where pCPP430 and pCPP450 overlap, therefore, was subcloned andits sequence was determined. This revealed four open reading frames,designated ORF A, ORF B, ORF C and hrpW (FIG. 1B). A putativeHrpL-dependent promoter (Bogdanove, et al., J. Bacteriol., 178:1720–30(1996) and Kim et al., J. Bacteriol., 179:1690–97 (1997), which arehereby incorporated by reference), CGGAACC-N₄-C-N₁₀-CCACTCAAT (SEQ. ID.No. 3), was found 58-bpi upstream of the hrpW start codon, suggestingthat the expression of hrpW is controlled by HrpL, an alternate sigmafactor (Wei, et al., J. Bacteriol., 177:6201–10 (1995), which is herebyincorporated by reference). hrpW in pCPP1232 (FIG. 1B) was expressedusing a T7 RNA polymerase/promoter system, and a specific protein bandwith an apparent molecular weight of ca. 60-kDa resulted (FIG. 2). Thisis larger than its expected size of 45-kDa. The same size band, however,was observed from the supernatant of E. amylovora (FIG. 4), indicatingthat the aberrant size of the protein is not a cloning artifact.

Example 11 Predicted Features of the hrpW Product

hrpW was deducted to encode a protein of 447-aa residues, which isacidic (p1–4.5), hydrophilic, rich in Gly, Ser, and Asn, low in Glu,Arg, Trp, and Tyr, and lacking in Cys (FIG. 3). These properties aresimilar to harpins, although the primary structure of HrpW seemed nothomologous to any of them. The sequence of HrpW suggests that theprotein is composed of two domains: the N-terminal Gly-and Ser-richdomain and the C-terminal domain homologous to PLs (see below). Abouttwo-thirds of Gly and Ser are located in the N-terminal region. The Glyand Ser content of the first 240-aa residues is 17.5% and 14.2%,respectively. The N-terminal region could be divided into fivesubregions, and contained two sequences (residues 40–59 and 131–145)that may form amphipathic α-helices. The first 39 residues of theN-terminus contains many Gly, Ser, Leu, and Asn, but few charged oraromatic amino acids. Similarly, the region that connects the twopotential α-helices has high Gly, Asn, and Gin content, but no aromaticresidues. Residues 146–232 contain several repeats ofSer/Thr-Pro/Ser/Thr-Pro/Ser/Thr, suggesting that this region might be alinker (Gilkes, et al., Microbiol. Rev., 55:303–15 (1991), which ishereby incorporated by reference).

Example 12 C-terminus of HrpW is Homologous to Pectate Lyases

Database searches using BLAST and FASTA algorithms (Altschul, et al., J.Mol. Biol., 215:403–10 (1990) and Pearson, et al., Proc. Natl., Acad.Sci. USA, 85:2444–48 (1988), which are hereby incorporated by reference)indicated that the C-terminal region of HrpW is homologous to PLA-D ofNectria haematococca mating type VI (Fusarium solani f. sp. pisi)(Gonzalez, et al., J. Bacteriol., 174:6343–49 (1992), Guo, et al., J.Bacteriol., 177: 7070–77 (1995), Guo, et al., Arch. Biochem. Biophys.,323:352–60 (1995), and Guo, et al., Arch. Biochem. Biophys., 332:305–12(1996), which are hereby incorporated by reference). BLAST P( ) valuesand FASTA E( ) values from runs with default parameters were 4.0e-14 to3.03-10 and 2.7e-08 to 1e-06, respectively. Based on BESTFIT alignments,HrpW was 27–33% identical to the fungal PLs and the Z-scores were 8.14to 13.3 Also, database search with PLs of N. haematococca showed thatthey are homologs of Pel-3 and PelB of Erwinia carotovora subsp.carotovora (Liu, et al., Appl. Env. Microbiol., 60:2545–52 (1994) andHeikinheimo, et al., Mol. Plant-Microbe Interact., 8:207–17 (1995),which are hereby incorporated by reference) (BLASTP P( ) values rangedfrom 9.0e-15 to 8.6e-10, and BESTFIT identities were 31–36%): Thesefungal PLs and E. carotovora Pel-3/PelB, together with HrpW, form aclass distinct from other PL families. From an alignment of theproteins, five highly conserved blocks were recognizable (FIG. 3). Theseven members share 20 identical residues of which five are Gly. The PHDalgorithm predicted &-sheets and loops for the PL-homology region ofHrpW, except for the sequence at residues 329–336 which has a propensityto form an α-helix (FIG. 3). Intriguingly, HrpW does not contain anyCys, which are conserved among PLs in the class. In addition, PLactivity of HrpW was not detected using the several tests described inthe materials and methods.

Example 13 Production and Secretion of HrpW are hrp-Dependent

An immunoblot with anti-HrpW antibodies detected HrpW only fromsupernatant preparations of E. amylovora Ea321 and Ea273, indicatingthat HrpW is efficiently secreted (FIG. 4). HrpW was not found inpreparations from hrpL mutants Ea321-K49 and Ea273-K49, demonstratingthat expression of hrpW is hrpL-dependent. In addition, HrpW either wasnot detected or restricted to the whole cell preparations of hrpsecretion mutants Ea321-G84 and Ea273-G73, respectively. Thus, secretionof HrpW is Hrp pathway-dependent. Anti-HrpW antibodies did not reactwith HrpW (FIG. 4, lane 2), suggesting structural differences betweenthe two elicitors.

Example 14 HrpW Induces Rapid Tissue Necrosis on Plants in a Heat-Stableand Protease-Sensitive Manner

From the predicted properties of HrpW, it is inferred to be an HRelicitor. To test this possibility, the partially purified protein wasinfiltrated into tobacco leaves. The infiltrated area began to collapseafter 8–12 hr, and typical tissue necrosis, indistinguishable from thatelicited by HrpN, developed 24–36 hr after inoculation (FIG. 5A, panel9). HrpW induced tissue necrosis in tobacco at concentrations of 1.1 μM(5 0 μg/ml). HrpW also caused necrosis in African violet, geranium,tomato, pepper, Kalanchoe diagremontiana, and Arabidopsis thaliana, butnot in soybean. A heat-treated preparation of HrpW still caused rapidnecrosis in tobacco leaves, indicating the heat-stable nature of theactivity (FIG. 5A, panel 2). On the other hand, treatment of HrpW with 3mg/ml protease (type XIV; Sigma, St. Louis, Mo.) for 1 hr destroyedHR-eliciting activity.

Example 15 Elicitation by HrpW Requires Plant Metabolism

A major question was whether the tissue necrosis caused by HrpW is dueto a mechanism comparable to harpins (He, et al., Cell, 73:1255–66(1993) and He, et al., Mol. Plant-Microbe Interact., 7:289–92 (1994),which are hereby incorporated by reference). Coinfiltration of HrpW CFEPwith the metabolic inhibitors cycloheximide, lanthanum chloride, orsodium vanadate (targets are 80S ribosome, Ca²⁺ channels,ATPases/Y-phosphateses, respectively) prevented the HR (FIG. 5B, panels3–5), like HrpN CFEP with the inhibitors (FIG. 5B, panels 7–8). Thisindicates that active plant metabolism is needed for the HrpW-inducedHR. Tobacco leaves infiltrated with PelE of E. chrysanthemi EC 16 alsoexhibited rapid tissue necrosis (FIG. 5B, panel 9). However, necrosiscaused by PelE occurred faster and the collapsed area was translucent,darker, softer, and easily crushed as compared to that elicited byharpins. In addition, PelE induced tissue necrosis irrespective of thepresence of inhibitors (FIG. 5B, panels 10–11).

Example 16 N-terminal Region is Sufficient for HR Elicitation

A fragment of hrpW encoding the N-terminal 226 residues, designatedHrpW(1–226), was constructed, and the production of HrpW(1–226) wasconfirmed. Typical HR developed 24–36 hr after infiltration ofHrpW(1–226) CFEP into tobacco leaves, though the activity was weakerthan that of full-length HrpW. That HrpW(1–226) are produced stably andelicits the HR independently of the C-terminal region support thetwo-domain structure of HrpW derived from the sequence data.

Example 17 hrpW is Conserved Among Strains of E. amylovora

The presence of hrpW in other bacteria was examined by Southernhybridization. Under high stringency conditions, single bands wereobserved for each of the ten strains of E. amylovora tested. The sizesof the restriction bands suggested three different groups. When lowstringency conditions were used, hybridizing bands were visible fromseveral other species of Erwinia such as E. carotovora, and E. salicis,and pCPP2157, a clone containing the hrp gene cluster of E. chrysanthemi(Bauer, et al., Mol. Plant-Microbe Interact., 8:484–91 (1995), which ishereby incorporated by reference) (FIG. 6).

Previously, at most only one harpin was known from each bacterium.However, E. amylovora encodes HrpW, a harpin distinct from the firstharpin described (HrpN; Wei et al., Science, 257:85–88 (1992), which ishereby incorporated by reference). Southern analysis suggests that hrpWexists in several other Erwinia species, suggesting a role for HrpW inpathogenesis. Furthermore, sequence comparison indicated that EXP-60(renamed to HrpW of P. syringae pv. tomato (Yuan, et al., J. Bacteriol.,178:6399–6402 (1996), which is hereby incorporated by reference) ishomologous to HrpW of E. amylorova.

Analysis suggests that HrpW is a multidomain protein comprised of theN-terminal Gly/Ser-rich domain and the C-terminal PL-homology domain.That HrpW has homology to PLs was surprising, because E. amylovora isbelieved to be non-pecolytic (Seemüller, et al., Phytopathology,66:433–36 (1976), which is hereby incorporated by reference). Besides,no pectic enzyme function has been suggested for harpins (Alfano, etal., Plant Cell, 8:1683–98 (1996), which is hereby incorporated byreference). Although PL activity was not detected, HrpW is not the firstPL homolog for which pectic enzyme activity function has not beendemonstrated. Some plant pollen proteins are PL homologs and pecticenzyme function has been suspected from the pollen-specific expressionof the encoding genes (Wing, et al., Plant Mol. Biol., 14:17–28 (1990),which is hereby incorporated by reference); however, they do not havedetectable enzyme activity (Dircks, et al., Plant Physiol. Biochem.,34:509–20 (1996), which is hereby incorporated by reference). HrpW maydiffer in substrate specificity from its homologs, or it may lack thepectic enzyme function like α-lactalbumins, which are homologs oflysozymes but do not have lysozyme function (McKenzie, et al., Adv.Prot. Chem., 41:173–315 (1991), which is hereby incorporated byreference). Alternatively, instead of lyase function, HrpW may have onlya pectic substance-binding function (Kurosky, et al., Proc. Natl., Acad.Sci. USA, 77:3388–92 (1980), which is hereby incorporated by reference).

HrpW is exceptional as a PL homolog in several respects. It does notposses the N-terminal signal peptide that is recognized by the Secmachinery; rather, it is secreted via a type III pathway. It does notcontain any Cys residues conserved in PLs, which may have structural andfunctional roles. Furthermore, HR elicitation by HrpW depends on plantmetabolism. Southern data suggests that E. caratovora has a hrpW homologdifferent from pel-3/pelB. Considering its dependence on the hrp systemand the lack of detectable PL activity, the role of HrpW in pathogenesismay not be for simple degradation of cell wall for utilization as acarbon source; rather, through possible pectic enzyme activity or cellwall-binding activity, it may assist the Hrp pilus (Roine, et al., Proc.Natl. Acad. Sci. USA, 94:3459–64 (1997), which is hereby incorporated byreference) or other pathogenicity/virulence/avirulence proteins ingetting through the cell wall.

Several evolutionary related groups of pectic enzymes are apparent basedon amino acid comparisons and structural analyses: i) a class called the“extracellular PL superfamily” (Henrissat, et al., Plant Physiol.,107:963–76 (1995), and references therein, which are hereby incorporatedby reference) that includes two PL families of plant pathogenicbacteria, Bacillus subtilis and some fungi, PelX of E. caratovora,pectin lyases, and plant pollen and style proteins, ii) a classcomprising periplasmic PLs of Yersinia pseudotuberculosis and E.caratovora (Hinton, et al., Mol. Microbiol., 3:1785–96 (1989), which ishereby incorporated by reference) and KdgC of E. chrysanthemi(Condemine, et al., Mol. Microbiol., 5:2191–2202 (1991), which is herebyincorporated by reference), iii) a class that consists of PLs of N.haematococca, and Pel-3/PelB of E. carotovora, iv) PelX and PelL of E.chrysanthemi (Alfano, et al., J. Bacteriol., 177:4553–56 (1995) andLojkowska, et al., Mol. Microbiol., 16:1183–95 (1995), which are herebyincorporated by reference), and v) recently reported PelZ of E.chrysanthemi and E. carotovora (Pissavin, et al., J. Bacteriol.,178:7187–96 (1996), which is hereby incorporated by reference). Thethird class of homology group has not yet been recognized as a proteinfamily, although members have been mentioned in the literature(Henrissat, et al., Plant Physiol., 107:963–76 (1995) and Liao, et al.,Mol. Plant-Microbe Interact., 9:14–21 (1996), which are herebyincorporated by reference). Therefore, this third class, to which HrpWof E. amylovora belongs, will be referred to as “class III pectatelyases” to differentiate it from the two earlier classes, which shouldbe called “class I” and “class II”. Members of the “class III PLs”appear to be widespread among plant pathogens. Besides HrpW in P.syringae pv. tomato, E. chrysanthemi has Pell which is very closelyrelated to Pel-3/PelB of E. carotovora.

It is enigmatic how harpins apparently heterogeneous in sequence andpossibly in structure can induce the same plant response. The“cell-killing” action of harpins appears not due to potential enzymaticor toxic function such as making pores in the cell membrane; HR activityis heat-stable (Wei, et al., Science, 257:85–88 (1992), He, et al.,Cell, 73:1255–66 (1993), and Arlat, et al., EMBO J., 13:543–53 (1994),which are hereby incorporated by reference), requires plant metabolism(He, et al., Cell, 73:1255–66 (1993), and He, et al., Mol. Plant-MicrobeInteract., 7:289–92 (1994), which are hereby incorporated by reference),and fragments can elicit the reaction (Arlat, et al., EMBO J., 13:543–53(1994), Alfano, et al., Mol. Microbial., 19:715–28 (1996), and Laby, etal., Molecular Studies on Interactions Between Erwinia amylovora and ItsHost and Non-Host Plants, Cornell University, Ithaca, N.Y. (1997), whichare hereby incorporated by reference). Avr proteins induce the HR onplants carrying corresponding resistance genes (Staskawicz, et al.,Science, 268:661–67 (1995), which is hereby incorporated by reference).It seems less probable that harpins evoke the HR by the same kind ofmechanism, although downstream signaling events could be shared.Possible signaling mechanisms that lead to the HR against harpins werediscussed by Novacky and colleagues (Hoyos, et al., Mol. Plant-MicrobeInteract., 9:608–16 (1996), which is hereby incorporated by reference).

It appears that Gly/Ser richness may be important for HR-elicitingfunction of harpins, because non-overlapping fragments that elicit theHR include Gly/Ser-rich regions (Arlat, et al., EMBO J., 13:543–53(1994), Alfano, et al., Mol. Microbial., 19:715–28 (1996), and Laby, etal., Molecular Studies on Interactions Between Erwinia amylovora and ItsHost and Non-Host Plants, Cornell University, Ithaca, N.Y. (1997), whichare hereby incorporated by reference). In support of the hypothesis, atruncated HrpW containing the N-terminal Gly/Ser-rich domain hasHR-eliciting ability. On the other hand, HR elicitation by fragments isweaker as compared to whole protein (Laby, et al., Molecular Studies onInteractions Between Erwinia amylovora and Its Host and Non-Host Plants,Cornell University, Ithaca, N.Y. (1997) and this work, which are herebyincorporated by reference) indicating that other part(s) of harpinscontribute to the full-strength HR. It will be of interest to determinewhether plant cell wall Gly-rich proteins (“GRPs”), the encoding genesof which are expressed during xylogenesis and after wounding or viralinfection (Showalter, A. M., Plant Cell, 5:9–23 (1993), which is herebyincorporated by reference), possess the ability to cause cell death.

Harpins appear to be targeted to outer parts of plant cells such as thecell wall. They can elicit the HR when exogenously applied to planttissue by infiltration. When harpins are added to cell-suspensionculture, K⁺ efflux and alkalinization of the medium, referred to asexchange reaction (“XR”), followed by cell death occurs (Wei, et al.,Science, 257:85–88 (1992) and Popham, et al., Physiol. Mol. PlantPathol., 47:39–50 (1995), which is hereby incorporated by reference).However, the XR does not occur in protoplast culture. In addition, HrpZantibodies localize HrpZ outside of plant cells and not in protoplasts,and the alkalinization and the localization is blocked by a chelatingagent that extracts Ca²⁺ and pectin (Hoyos, et al., Mol. Plant-MicrobeInteract., 9:608–16 (1996), which is hereby incorporated by reference).The homology of HrpW to PLs is consistent with a model in which the siteof harpin action is the plant cell wall.

Type III systems of animal pathogens secrete many proteins involved inpathogenesis (for example, see Cornelis, et al., Mol. Microbiol.,23:861–67 (1997), which is hereby incorporated by reference). Untilrecently, however, only harpins have been shown to be delivered by thetype III machinery of plant pathogens. Recent evidence suggests thatmultiple proteins are secreted through the Hrp pathway, and that severalAvr proteins are transferred directly into the plant cell by way of theHrp secretion machinery (Gopalan, et al., Plant Cell, 8:1095–1105(1996), Leister, et al., Proc. Natl. Acad. Sci. USA,93:15497–15502(1996), Scofield, et al., Science 274:2063–65 (1996),Tang, et al., Science, 274:2060–63 (1996), and Van Den Ackerveken, etal., Cell, 87:1307–16 (1996), which are hereby incorporated byreference).

It is interesting that hrpW is flanked by dspE and ORF B (FIG. 1B),which are homologs of avrE of P. syringae and avrRxv of X. campestrispv. vesicatoria, respectively. The linkage of harpin genes and homologsof non-host avr genes provides a hint of relationships between them inpathogenesis. Harpins might in reality be a class of Avr proteins, orAvr proteins may be actually virulence proteins. PopA of P. solanacearumGMI1000 elicits the HR only in resistant petunia lines (Arlat, et al.,EMBO J., 13:543–53 (1994), which is hereby incorporated by reference).Also, expression of the Avr phenotype is controlled by the hrp system,and some avr genes possess virulence or pathogenicity functions (Dangl,Curr. Top. Microbiol. Immunol., 192:99–118 (1994), which is herebyincorporated by reference). Indeed, dspE is a pathogenicity factor.Thus, the region of the E. amylovora genome where harpin genes and avrhomologs reside may constitute an arsenal for proteins used to bombarddifferent parts of the host cell. Elucidating their specific targets andeffects in the HR and pathogenesis will be pivotal to understandmechanisms of plant-bacterial interactions.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An isolated DNA molecule encoding a hypersensitive response elicitingprotein or polypeptide, wherein the isolated DNA molecule is selectedfrom the group consisting of (a) a DNA molecule comprising SEQ ID NO: 1and (b) a DNA molecule encoding a protein comprising SEQ ID NO: 2, or anisolated DNA molecule complementary to DNA molecules (a) or (b).
 2. Theisolated DNA molecule according to claim 1, wherein said DNA molecule isa DNA molecule comprising SEQ ID NO:
 1. 3. The isolated DNA moleculeaccording to claim 1, wherein said DNA molecule is a DNA moleculeencoding a protein comprising SEQ ID NO:
 2. 4. The isolated DNA moleculeaccording to claim 1, wherein said DNA molecule is a DNA moleculecomplementary to DNA molecules (a) or (b).
 5. An expression vectorcomprising the DNA molecule of claim 1 and a promoter operably coupledto the DNA molecule.
 6. The expression vector according to claim 5,wherein the DNA molecule is in sense orientation relative to thepromoter.
 7. A host cell transformed with the DNA molecule of claim 1.8. The host cell according to claim 7, wherein the host cell is a plantcell or a bacterial cell.
 9. The host cell according to claim 7, whereinthe DNA molecule is operably coupled to a promoter comprised within anexpression vector.
 10. An isolated DNA molecule of an Erwinia pathogen,wherein the isolated DNA molecule both encodes a polypeptide thatelicits a hypersensitive response in non-host plants, and hybridizes toa DNA molecule comprising the complement of SEQ ID NO: 1 underhybridization conditions comprising hybridization at 50° C. for 24 hoursin a solution that comprises 6×SSC and 0.5% SDS, followed by washconditions comprising a first wash at 45° C. in a solution thatcomprises 2×SSC and a second wash at 45° C. in a solution that comprises0.1×SSC.
 11. The isolated DNA molecule according to claim 10 wherein theencoded polypeptide contains an N-terminal hypersensitive responseeliciting domain and a C-terminal pectate lyase-homologous domain thatlacks pectate lyase activity.
 12. The isolated DNA molecule according toclaim 10 wherein the encoded polypeptide is acidic, hydrophilic,protease sensitive, and lacks cysteine.
 13. The isolated DNA moleculeaccording to claim 10 wherein the Erwinia pathogen is selected from thegroup of E. amylovora, E. carotovora, E. salicis, and E. chrysanthemi.14. An expression vector comprising the DNA molecule of claim 10 and apromoter operably coupled to the DNA molecule.
 15. The expression vectoraccording to claim 14, wherein the DNA molecule is in sense orientationrelative to the promoter.
 16. A host cell transformed with the DNAmolecule of claim
 10. 17. The host cell according to claim 16, whereinthe host cell is a plant cell or a bacterial cell.
 18. The host cellaccording to claim 16, wherein the DNA molecule is operably coupled to apromoter comprised within an expression vector.